Composition of a n-carboxymethylated tetraazacyclododecane chelating agent, a paramagnetic metal and excess calcium ions for MRI

ABSTRACT

Magnetic resonance imaging contrast media used to affect the relaxation times of atoms in body tissues undergoing NMR diagnosis are disclosed. Such media comprise (1) a paramagnetic, physiologically compatible complex of (a) a chelant consisting of a N-carboxy methylated tetraazacyclododecane, and (b) a paramagnetic ion of a lanthanide element of atomic numbers 59-70, or of a transition metal of atomic numbers 21-29, 42 or 44, or a physiologically compatible salt of such a complex, and (2) a toxicity-reducing amount of labile calcium ions. The labile calcium ions can be derived from a source other than a salt of said chelant, e.g. an inorganic or organic calcium salt. NMR image-enhancing compositions comprising NMR image-enhancing effective amounts of said contrast media and pharmaceutically acceptable diluents are also disclosed.

This is a division of application Ser. No. 07/057,709, filed Jun. 15,1987, which was a continuation-in-part of U.S. patent application Ser.No. 893,136, filed Aug. 4, 1986, abandoned, and of U.S. patentapplication Ser. No. 900,930, filed Aug. 27, 1986, abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to improvements in the enhancing ofnuclear magnetic resonance (NMR) imaging of animal tissues, especiallycardiac and liver tissues.

X-rays have long been used to produce images of animal tissue, e.g., theinternal organs of a patient, the patient being positioned between asource of X-rays and a film sensitive to the rays. Where organsinterfere with the passage of the rays, the film is less exposed and theresulting developed film is indicative of the state of the organ.

More recently, another imaging technique has been developed, viz.nuclear magnetic resonance. This avoids the harmful effects sometimesattending X-ray exposure. For improved imaging with X-rays, patientshave been given enhancers prior to imaging, either orally orparenterally. After a predetermined time interval for distribution ofthe enhancer through the patient, the image is taken. To obtain a goodimage it is desirable that the time after the taking of enhancer be keptto a minimum. On the other hand there is a decrease in effectivenesswith time, so desirably the decay should be relatively slow so as toprovide a substantial time interval during which imaging can be done.The present invention relates to enhancers for NMR imaging.

In the NMR imaging process, protons in the water of the body relax viatwo mechanisms referred to as T₁ and T₂. The rate at which therelaxation process occurs may be altered for some water molecules bygiving values that contrast with the norm.

Chemicals that enhance NMR images, referred to as contrast agents, aregenerally paramagnetic in nature. These may be organic free radicals ortransition/lanthanide metals which have from one to seven unpairedelectrons.

A necessary prerequisite of any ligand that chelates (binds) a metal toform a contrast agent is that it be stable so as to prevent the loss ofthe metal and its subsequent accumulation in the body. Otherconsiderations include an ability to reversibly bind water, which inturn increases its contrastability and decreases the dose levelrequired. This ability is clearly important since the interactionbetween any two nuclear spins through space decreases at a rate equal tothe reciprocal of the distance raised to the sixth power.

U.S. Pat. No. 4,647,447 discloses use of an NMR image enhancerconsisting of the salt of an anion of a complexing acid and aparamagnetic metal ion. A preferred embodiment is the gadolinium chelateof diethylenetriaminepentaacetic acid (Gd DTPA). From the data reportedtherein these appear to perform well. However, this compound is rapidlyexcreted by the kidneys, making the timing of the injection extremelycritical. Furthermore, there is virtually no uptake by any solid organ,such as the heart, pancreas or liver.

However, while a number of gadolinium contrast agents are known to workwell, there remains the possibility that small amounts of freelanthanides are being released, by decomposition of the agent, into thebody. Not being a naturally existing metal in the body, little is knownabout long term effects.

It is accordingly an object of the present invention to providealternative image enhancers which avoid one or more of theaforementioned disadvantages.

It is another object of the invention to provide an NMR-image enhancerwhich does not release lanthanides into the body.

SUMMARY OF THE INVENTION

These and other objects and advantages are realized in accordance withone aspect of the present invention pursuant to which there is provideda calcium or magnesium salt of a paramagnetic, physiologicallycompatible salt of a physiologically compatible chelate complex of anion of a lanthanide element of atomic numbers 57-70, or of a transitionmetal of atomic numbers 21-29, 42, or 44.

Advantageously, the salt of the chelate complex is of the formula I orII ##STR1## or

    N(CH.sub.2 X).sub.3,                                       (II)

wherein

X is --COOY, --PO₃ HY or --CONHOY;

Y is a hydrogen atom, a metal ion equivalent or a physiologicallybiocompatible cation of an inorganic or organic base or amino acid;

A is --CHR₂ --CHR₃ --, --CH₂ --CH₂ --(ZCH₂ --CH₂)_(m) --, ##STR2## eachR₁ is a hydrogen atom or methyl;

R₂ and R₃ together represent a trimethylene group or a tetramethylenegroup or individually are hydrogen, C₁₋₈ -alkyl, phenyl or benzyl,

W is --N═N--, --NHCOCH₂ -- or --NHCS--;

m is the number 1, 2 or 3,

z is an oxygen atom, a sulfur atom, NCH₂ X, or NCH₂ OH₂ R₄,

R₄ is C₁₋₈ -alkyl,

V is one of the X groups or is --CH₂ OH, or --CONH(CH₂)_(n) X,

n is a number from 1 to 12;

if R₁, R₂ and R₃ are hydrogen atoms, both V's together are the group##STR3##

w is a number 1, 2 or 3;

provided that at least two of the substituents Y are metal ionequivalents of an element with an atomic number of 21 to 29, 42, 44 or57 to 83,

and at least one is calcium or magnesium.

Alternatively the calcium or magnesium salt may be a complex of an ionand a ligand, the complexed ion being an ion of a lanthanide element ofatomic numbers 57-70, or of a transition metal of atomic numbers 21-29,42, or 44; and the ligand being that of a calcium or magnesium salt ofan organic complexing agent which is acyclic or cyclic and containsorganic nitrogen, phosphorus, oxygen or sulfur. In this embodiment,advantageously the complexing agent which forms a

(a) an aminopolycarboxylic acid which is nitrilotriacetic acid,N-hydroxyethyl--N,N',N'',N'''-ethylenediaminetriacetic acid,N,N,N',N'',N'''-diethylenetriaminepentaacetic acid orN-hydroxethyliminodiacetic acid;

(b) of the formula ##STR4## wherein R₁ and R.sub..12 ' are identical ordifferent and each is hydrogen or alkyl of 1-4 carbon atoms and p is aninteger of 0-4; or

(c) an aminopolycarboxylic acid of the formula ##STR5## wherein

m is an integer of 1 to 4,

n is an integer of 0 to 2, and

R₅ is C₄₋₁₂ -alkyl, C₄₋₁₂ -alkenyl, C₄₋₁₂ -cycloalkyl, C₄₋₁₂-cycloalkenyl, C₇₋₁₂ -hydrocarbon aralkyl, C₈₋₁₂ -hydrocarbon alkeynl,C₆₋₁₂ --hydrocarbon aryl or --CH₂ COOH.

Such salts are especially useful in the NMR diagnosis of patients towhom they are administered followed by imaging.

The acid moiety of the chelate is advantageously carboxy and phosphono,sulpho being less advantageous. The acid groups are joined to the aminonitrogen by an alkyl, i.e. alkylene, radical of up to 4 carbon atoms.Preferably they are acetic acid radicals, i.e., di-carboxymethylaminoradicals, or phosphonic acid radicals as in U.S. Pat. No. 3,738,937.

Preferably there are two amino groups on adjacent carbon atoms andpreferably still they are in the trans configuration, e.g.,trans-N,N,N',N'-tetracarboxymethyl-1,2-diaminocyclohexane.

If desired, up to two of the carboxylic acid groups may be reacted toform an amide, a lower alkyl ester and/or an anhydride.

The polyvalent paramagnetic metal may be any of those heretofore used inNMR image enhancement, e.g., iron, chromium, cobalt, nickel, neodynium,promethium, samarium, europium, terbium, dysprosium, holmium, erbium,thorium, ytterbium and lutetium Preferably, however, the metal is iron,manganese, or gadolinium.

The metal containing complex is made by adding the cyclic compound towater and adding four mole equivalents of an alkali such as sodiumhydroxide or N-methyl-d-glucamine to dissolve the compound. A 1 molarequivalent of manganese chloride or gadolinium chloride is nowintroduced into the solution. As a result of the chelate formation, thepH of the solution drops to about 5. When manganese chloride is used,rigorous degassing of all water used and compound formation under aninert nitrogen blanket combine to prevent the formation of oxideproducts during the course of the reaction. The final pH is adjusted tobetween 5 and 8 and the solution is passed through a 0.2 micron filterfor sterilization.

The osmolarity of the resulting solution can be lowered to aphysiologically acceptable value by removal of the unnecessary butphysiologically acceptable sodium chloride by-product. This can beachieved by crystallization, filtering, dialysis or ion exchange.

The superiority of ring-based contrast agents over other contrast agentswhich have straight alkane chain backbones, e.g., EDTA(ethylenediaminetetraacetic acid) or DTPA (diethylenetriaminepentaaceticacid) apparently resides in the cyclohexane backbone which imparts morerigidity to the molecule and sterically hinders the coordination ofwater into the nitrogen-metal bond position. While EDTA divalent metalcompounds tend to first break the metal nitrogen bonds by watercoordination, the instant system loses the oxygen donors first. This isreflected in the proton nuclear magnetic resonance spectrum of therespective molecules. For example, the manganese salt oftrans-N,N,N',N'-tetracarboxymethyl-1,2-diaminocyclohexane (DCTA) has amanganese-nitrogen bond which is considerably more stable than its EDTAanalogue. This is reflected in the stability constant (binding ability)towards manganese which is several thousand times better for DCTA thanthe EDTA chelate. Even though the stability constant of the novelgadolinium complex is approximately the same as the stability constantof Gd DTPA, it is important to note that the novel complex is atetraacidic ligand while DTPA is pentaacidic. Consequently, inner spherewater coordination is greater and the corresponding relaxation valuesare considerably better. This improvement allows a decrease in dosageand hence a decreased possible toxicity through degradation and releaseof free gadolinium.

The addition of calcium or magnesium to the complexes reduces theirtoxicity. The calcium or magnesium should be present in about 0.1 to200% and preferably about 10 to 100% based on the moles of paramagneticpolyvalent metal. It can be an inorganic salt such as the chloride orsulfate, but organic salts, e.g., the gluconate, lactate, ascorbate,etc., are preferred.

A calcium or magnesium salt can simply be added to the complex insolution and so administered or the solution can be dried and the drymaterial later redissolved.

The addition of the calcium or magnesium to the chelate saltsurprisingly serves to increase the safety, i.e., to raise the LD₅₀based on the amount of paramagnetic polyvalent metal present.

For example, the MnEDTP chelate without calcium has an LD₅₀ of 200umol/kg, a toxic level. The LD₅₀ of the same complex into which 40 mol %of calcium has been incorporated, via calcium gluconate, is in excess of850 umol/kg, a relatively safe level for human use.

In accordance with another aspect of the invention the acid group is aphosphono moiety. This aspect is applicable even to compounds which arenot cyclic, e.g., linear alkylene polyamines such aspoly(nitrogen-substituted) phosphonoalkyl alkylenepolyamines, and tocompositions not containing a calcium or magnesium salt.

As the poly-phosphono alkylated alkylene polyamine there are preferablyemployed compounds wherein the alkyl and alkylene radicals each containup to four carbon atoms. The alkylenepolyamine could bediethylenetriamine, for example, but ethylenediamine is preferred.Advantageously the phosphono groups are joined to the nitrogen atomsthrough a methyl group, i.e., actually a methylene group. Each phosphonogroup has two acid moieties so in a compound having four nitrogen atomsthere are eight acid moieties available for complexing.

If desired, up to half of those acid moieties can be bound as salts withnon-paramagnetic cations, e.g., alkali metal, alkaline earth metal orammonium salts, or they may be combined as lower alkyl esters, amidesand/or anhydrides. The calcium added as the calcium salt has abeneficial effect even beyond that realized if the acid moieties of thepolyphosphono alkylated alkylene polyamine are already partially incalcium salt form, for example.

One preferred complexing or chelating agent of this type isN,N,N',N'-tetraphosphonomethylethylenediamine (EDTP) of the structuralformula ##STR6## which is commercially available in the form of itssodium salt and free acid.

While lanthanides and particularly gadolinium are highly paramagneticand useful in accordance with the invention, it is surprising that otherless paramagnetic metals perform well, e.g., iron, manganese, copper,cobalt, chromium and nickel.

The complex can be prepared by dissolving a salt of EDTP in water orother solvent and adding a salt of the desired metal, e.g., manganesechloride, in from about half to twice the stoichiometric amount.Additional salts, such as calcium chloride, can be added to tie upadditional binding sites in the compound. The solution can then bedialyzed or ion exchanged to remove chloride ions or an alkali such asNaOH can be added to neutralize the chloride ions, the by-product NaClbeing removed or left in solution since it is physiologicallyacceptable.

The Mn-EDTP complex distributes substantially to the following organs:liver, heart, kidneys, spleen, pancreas, bladder, stomach, small andlarge intestines.

As noted, manganese is the preferred metal, but other polyvalentparamagnetic metals may be used, e.g., iron, chromium, cobalt, nickel,copper, and the like. The preferred lanthanide is gadolinium, but otherssuch as lanthanum, cerium, praseodymium, neodymium, promethium,samarium, europium, terbium, dysprosium, holmium, erbium, thulium,ytterbium and lutetium may also be used.

This invention may be used in conjunction with any magnetic resonancemachine currently available and is compatible with any of the currentknown imaging techniques, e.g., a machine such as that of Siemens AG ofErlanger, Federal Republic of Germany.

Further details of imaging systems are described in the prior art, e.g.,"NMR A Primer for Medical Imaging" by Wolf and Popp Slack Book Division(ISBN 0-943432-19-7) and Scientific American, May 1982, pages 78-88.

The solution of complex may be sterilized and made up into ampules ormay be lyophilized into a powder for dissolution when ready to be used.The solution may be mixed with conventional additives such as salinesolution, albumin, buffers and the like. If desired, ampules may be madeup containing lyophilized powder of the complex in one compartment and asolution of additives in another separated from the first by a frangiblebarrier. When ready to use, the barrier is broken and the ampule shakento form a solution suitable for use.

Immediately prior to actual administration of the contrast agent, thereconstituted solution is further diluted by addition of a suitablediluent such as:

Ringer's Injection, USP

Sodium Chloride Injection, USP

Dextrose Injection, USP (5 percent Dextrose in sterile water)

Dextrose Sodium Chloride Injection, USP (5 percent Dextrose in SodiumChloride)

Lactated Ringer's Injection, USP

Protein Hydrolysate Injection Low Sodium, USP 5 percent 5 percent withDextrose 5 percent 5 percent with Invert Sugar 10 percent

Water for Injection, USP

The manner and dosage of administration and the manner of scanning aresubstantially the same as in the prior art. With solutions containingabout 50 to 500 mmoles of the complex per liter, sufficient solutionshould be administered orally or parenterally to provide about 1 to 1 00umols/kg, corresponding to about 1 to 20 mmol for an adult humanpatient.

For smaller patients or animals, the dosage should be variedaccordingly. The particular complex and organ to be imaged willdetermine the waiting period between administration and imaging. It willgenerally be at least about 15 minutes but less than about an hour.During the first few hours the complex is execreted by the liver intothe bile.

The invention will be further described in the following illustrativeexamples wherein all parts are by weight unless otherwise expressed.

EXAMPLE I

Synthesis of DCTP(trans-1,2-diaminocyclohexane-N,N,N,N-tetramethylenephosphonic acidhydrate).

28.5 g (0.25 mole) of trans-1,2-diaminocyclohexane and 82 g (1 mole) ofphosphorous acid are dissolved in 140 ml of concentrated hydrochloricacid. The solution is heated to reflux (110° C.) and 162 g (2.1 moles)of formalin (40% aqueous solution of formaldehyde) are added over thecourse of 90 minutes. The temperature drops to 94° C. and the reactionis maintained at this temperature for 5 hours and then allowed to coolto 25° C. overnight. Crystallization is initiated via scratching thewalls of the flask. After standing overnight the precipitated product isisolated via filtration and washed with acetone (3×100 ml). The DCTP isrecrystallized from a minimum of water, isolated by filtration, washedwith acetone and air-dried. 64 g (52% yield) of pure product areobtained.

Characterization of DCTP

The melting point is 228°-232° C. (decomposition) with slight darkeningobserved above 220° C.

The positive ion mass spectrum shows a parent ion at 491 mass units(theoretical: 491). Elemental analysis for DCTP H₂ O (C₁₀ H₂₈ N₂ O₁₃ P₄; Calculated: C, 23.63; H, 5.55; N, 5.51; P, 24.38. Found: C, 23.87; H,5.41; N, 5.48; p, 24.49. Water, 3.71% by Karl-Fischer titration.

Spectrophotometric complexation analysis of DCTP with standardizedcopper chloride yields percentages of 100.1, 100.6 and 101.2 (average100.6) assuming a molecular weight of DCTP.H₂ O of 508.22.

Nuclear Maqnetic Resonance Spectra of DCTP

The proton (400.13 MHz), carbon (100.61 MHz) and phosphorous (161.94HMz) NMR spectra oftrans-1,2-diaminocyclohexane-N,N,N,N-tetramethylenephosphonic acid indimethyl sulfoxide-d6 do not provide structural and peak assignmentsthrough standard NMR techniques. Because of the number of overlappingpeaks, 2-dimensional 1H-13C chemical shift correlation NMR techniquesare required to make unequivocal peak assignments. The 2D NMR resultsand analysis of a molecular model indicate an axis of symmetry creatingtwo sets of non-equivalent phosphorous atoms and diasterotopic protonson the methylene carbons adjacent to the phosphorous atoms. The fourmethylene units create two sets of chemically non-equivalent nuclei. TheNMR peak assignments are as follows: 13C (ppm relative to TMS): 63.2(singlet, methine of cyclohexyl), 50.72 (doublet, Jcp=145.7 Hz,methylene set A of phosphonate), 47.10 (doublet, Jcp=140.4 Hz, methyleneset B of phosphonate), 23.9 (singlet, beta-methylene of cyclohexyl),22.9 (singlet, gamma-methylene of cyclohexyl). 1H (ppm relative to TMS):8.28 (P-OH), 3.55 (methine of cyclohexyl), 3.50, 3.31, 3.27, 2.88(methylene of phosphonate), 1.72, 1.16 (beta-methylene of cyclohexyl),2.10, 1.26 (gamma-methylene of cyclohexyl).

31P (ppm relative to H3PO4): -19.2, -19.8

The NMR results indicate that the DCTP ligand is relatively rigid on theNMR time-scale; in fact no interconversion is observed up to 60° C. Thisis in contrast to DCTA, the acetic acid analogue, which is rapidlyinterconverting on the NMR time-scale at 25° C.

EXAMPLE 2

Formation of Calcium Salt of Manganese Complex of DCTA and DCTP

a) To 60 ml of degassed water, 1.6 g (0.04 mole) of sodium hydroxide isadded. After the alkali is dissolved, 3.6436 g (0.01 mole) oftrans-N,N,N',N'-tetracarboxymethyl-1,2 diaminocyclohexane monohydrate(Aldrich Chemical Co., Milwaukee, Wis.) is added to the stirringsolution. 1.979 g (0.01 mole) of manganese chloride tetrahydrate isdissolved in 10 ml of degassed water and is added dropwise to theprevious solution. After 30 minutes of stirring, 0.1 mole equivalent ofcalcium chloride is added to the mixture. The pH of the solution isadjusted to 6.5, and water added to bring the final volume to 100 ml,resulting in a final concentration of 100 mM. The clear or faint yellowsolution is filtered through a 0.2 micron filter for sterilization.

b) The calcium salt of the maganese complex oftrans-1,2-diaminocyclohexane-N,N,N',N'-tetramethylene phosphonic acid(DCTP) is prepared from the product of Example 1 in a manner analogousto (a).

c) Relaxitivities of protons present in water and plasma exposed to thecomplexes of (a) and (b) (at 10 mHz) (37° C.) in milliseconds:

                  TABLE 1                                                         ______________________________________                                        Molar     T.sub.1    T.sub.2  T.sub.1 T.sub.2                                 Concentration                                                                           Water      Water    Plasma  Plasma                                  (moles/liter)                                                                           (a)     (b)    (a)  (b) (a)  (b)  (a)  (b)                          ______________________________________                                          1 × 10.sup.-2                                                                    32      16    22    8   25   15  50.5 10                             2 × 10.sup.-3                                                                    55      28    43   20   39   34  33.3 27                            2.5 × 10.sup.-3                                                                   95      54    69   36   74   51  16.9 45                           1.25 × 10.sup.-3                                                                  171      88    126  69  121   91   9.7 74                           6.25 × 10.sup.-4                                                                  322     172             223  142                                    3.12 × 10.sup.-4                                                                  599     310             336  212                                    1.56 × 10.sup.-4                                                                  971     555             513  269                                    7.80 × 10.sup.-5                                                                  1390    987             765  372                                    ______________________________________                                    

d) LD₅₀ values for 40 mice with the complex of (a):

                  TABLE 2                                                         ______________________________________                                        Dose (mmole/kg)                                                                             Sex      Fatalities                                                                             Survivors                                     ______________________________________                                        1.5           Male     0        5                                             1.5           Female   0        5                                             2.5           Male     1        4                                             2.5           Female   0        5                                             4.5           Male     2        3                                             4.5           Female   3        2                                             5.5           Male     4        1                                             5.5           Female   3        2                                             ______________________________________                                    

The LD₅₀ for (a) was determined to be 4.9 mmol/kg with a 95% confidencerange between 4.1 and 5.9 mmol/kg. The LD₅₀ for (b) is much lower at 0.2mmol/kg.

e) Organ distribution of (a) and (b) in male rabbits: The rabbits weresacrificed at 69 minutes post injection for (a) and 15 minutespost-injection for (b) and the proton relaxation values measured inmilliseconds, in vitro at 10 mHz, for each of the various organs.

                  TABLE 3                                                         ______________________________________                                                Normal                                                                        Values  (a)          (b)                                              Tissue    T.sub.1                                                                              T.sub.2                                                                              T.sub.1                                                                              T.sub.2                                                                             T.sub.1                                                                             T.sub.2                            ______________________________________                                        Brain     NA     NA     637    82    537   85                                 Heart     504    70     367    518   191   40                                 Lung      595    112    472    71    323   84                                 Fat       171    154    176    113   157   95                                 Skeletal Musc                                                                           423    47     539    62    395   34                                 Renal Cortex                                                                            338    85     123    42    109   51                                 Renal Medulla                                                                           672    149    232    71    103   47                                 Liver     252    64     182/137                                                                              28/37 82/66 27/24                              Pancreas  464    86     201    49    NA    NA                                 Stomach   349    69     226    52    199   42                                 Small lntest                                                                            352    79     115    46    269   60                                 Large Intest                                                                            349    77     219    44    248   58                                 Testis    NA     NA     623    123   294   79                                 Urine     NA     NA      17    11    NA    NA                                 ______________________________________                                         NA = Not Available                                                       

EXAMPLE 3

Formation of Calcium Salt of Gadolinium Complex of DCTA and DCTP

a) 18.218 g (0.05 mole) of trans-N,N,N',N'-tetra-carboxymethyl-1,2diaminocyclohexane is added to 100 ml of water and 8 g (0.2 mole) ofsodium hydroxide is added. 18.585 g (0.05 mole) of gadolinium chlorideis then added slowly while stirring. The solution is then stirred for anadditional 30 minutes A 0.1 molar equivalent of calcium chloride isadded at this point and the pH of the solution adjusted to 6.5. Thevolume of the solution is brought to 200 ml resulting in a finalconcentration of 250 mM. The solution is sterilized by passing through a0.2 micron filter.

b) The calcium salt of the gadolinium complex oftrans-1,2-diamino-cyclohexane-N,N,N',N'-tetramethylene phosphonic acidis prepared from the product of Example 1 in a manner analogous to (1).

c) Relaxivities of protons present in water and plasma exposed to (a)and (b) at 10 mHz (37° C.) in milliseconds:

                  TABLE 4                                                         ______________________________________                                        Molar     T.sub.1   T.sub.2   T.sub.1 T.sub.2                                 Concentration                                                                           Water     Water     Plasma  Plasma                                  (moles/liter)                                                                           (a)    (b)    (a)  (b)  (a)  (b)  (a)  (b)                          ______________________________________                                          1 × 10.sup.-2                                                                    22    15     14    8    25  14   20    7                             5 × 10.sup.-3                                                                    29    25     25   17    39  20   30   16                            2.5 × 10.sup.-3                                                                   55    49     47   35    74  36   59   26                           1.25 × 10.sup.-3                                                                  104    70     89   65   121  60   103  42                           6.25 × 10.sup.-4                                                                  183    126    161  114  223  95                                     3.12 × 10.sup.-4                                                                  367    257              336  149                                    1.56 × 10.sup.-4                                                                  562    468              513  263                                    7.80 × 10.sup.-5                                                                  983    762              765  447                                    ______________________________________                                    

d) For comparison purposes and to highlight the superior performance ofthe invention, there follows a table of relaxation values for water andplasma using the N-methyl glucamine salt of Gd DTPA:

                  TABLE 5                                                         ______________________________________                                        Molar Concentration                                                                           Water            Plasma                                       moles/liter     T.sub.1 T.sub.2  T.sub.1                                                                            T.sub.2                                 ______________________________________                                        6.25 × 10-3                                                                              40     35        39  31                                      3.13 × 10-3                                                                              83     76        69  61                                      1.56 × 10-3                                                                             163     155      134  116                                     7.81 × 10-4                                                                             309              240                                          3.91 × 10-4                                                                             582              405                                          1.95 × 10-4                                                                             1015             636                                          9.77 × 10-5                877                                          ______________________________________                                    

It is noted that the relaxation times in Table 1 with the novelmanganese complexes are approximately the same as the gadolinium saltsin Table 5, even though Table 1 employs a metal with two less unpairedelectrons and which is naturally occurring in the body. The gadoliniumsalts of this invention in Table 4 are still superior.

EXAMPLE 4

Preparation of 100 mM manganese EDTP Complex Containing 40 mM Calcium

(1) To 300 ml of water containing 0.2 mol of sodium hydroxide, 21.81 g(0.05 mol) of N,N,N',N'-tetraphosphonomethyleneethylenediamine (referredto as EDTP) is added. The mixture is stirred with a magnetic stirreruntil a clear solution is obtained. The pH of the resulting solution isapproximately 5.8.

(2) 9.90 g (0.05 mol) of manganese chloride tetrahydrate is dissolved inapproximately 15 ml of water and added to the stirring mixture. Aprecipitate is developed which dissolves on further stirring.

(3) 10 ml of 5M solution of sodium hydroxide is added to the stirringmixture to bring the pH to 5.8.

(4) 2.94 g (0.02 mol) of calcium chloride is added to the mixture. Aprecipitate that develops dissolves after about 15 minutes of stirring,and the pH drops to 5.6.

(5) The pH is brought back to 5.8 with a solution of 5M sodiumhydroxide.

(6) The solution is then brought to a final volume of 500 ml resultingin a concentration of 100 mM for the Mn-EDTP complex and 40 mM forcalcium.

(7) The solution is now filtered through 0.2 um filters and stored invials with butyl rubber stoppers.

The solution is then added to water and to human plasma in varyingamounts and the relaxivities measured in conventional manner forcomparison with those for the gadolinium complex of the2-N-methylgulcamine salt of diethylenetriaminepentaacetic acid shown inTable 5, supra.

The following results are obtained, low values for both T₁ (transverserelaxation mechanism) and T₂ (longitudinal relaxation mechanism) beingpreferred:

                  TABLE 6                                                         ______________________________________                                        Relaxivity of the Compound in Water and in Human                              Plasma in Milliseconds at 10 MHz (37° C.)                              Concentration                                                                              Water             Plasma                                         molar        T.sub.1 T.sub.2   T.sub.1                                                                            T.sub.2                                   ______________________________________                                          1 × 10.sup.-2                                                                       31     31         18  13                                          5 × 10.sup.-3                                                                       41     37         31  24                                         2.5 × 10.sup.-3                                                                      83     74         50  38                                        1.25 × 10.sup.-3                                                                     159     123        85  61                                        6.25 × 10.sup.-4                                                                     298               112  87                                        3.125 × 10.sup.-4                                                                    537               160  116                                       1.56 × 10.sup.-4                                                                     884               253  160                                       7.81 × 10.sup.-5                                                                     1326              353                                            3.91 × 10.sup.-5         478                                            1.95 × 10.sup.-5         585                                            9.77 × 10.sup.-6         653                                            4.88 × 10.sup.-6         797                                            ______________________________________                                    

The relaxivity of the Mn-EDTP-Ca is clearly superior to Gd DTPA. This isespecially evident in the T₁ values in plasma. For example, at aconcentration of 9.77×10⁻⁶ M, the value for Mn-EDTP-Ca complex is 653milliseconds; for Gd DTPA at a 10-fold higher concentration (9.77×10⁻⁵M) it is 877 msec, i.e., still higher.

EXAMPLE 5

Pharmacokinetics of the Compound of Example 4 in a Pure Breed Beagle Dog

Male dogs are injected with the solution of Example 4 and the comparisoncompound at 350 umol/kg. Blood is drawn at the indicated times. Theplasmas are separated and the T₁ relaxivities in milliseconds measured.

                  TABLE 7                                                         ______________________________________                                                      T.sub.1     T.sub.1                                             Time, Min.    Mn-EDTP-Ca  Gd DTPA                                             ______________________________________                                        Pre-inj       1102        1427                                                10             90         440                                                 20            108         444                                                 30            113         551                                                 45            153         580                                                 60            222         687                                                 90            404         860                                                 180           777         1282                                                360           968                                                             ______________________________________                                    

Plasma clearance of Gd DTPA is much faster than the Mn-EDTP-Ca Complex.By 180 minutes post-injection, most of the Gd DTPA is cleared from theplasma. Mn-EDTP-Ca is not cleared until 360 minutes post-injection. Thisgives Mn-EDTP-Ca a larger time "window" for imaging.

EXAMPLE 6

Organ Distribution of the Compound of Example 4 in Male Rabbits

The compound is injected into male rabbits at 50 umol/kg. The rabbitsare sacrificed at 15 minutes post injection and the T₁ relaxivity ofinternal organs measured in vitro at 5 MHz (milliseconds). The resultsare as follows:

                  TABLE 8                                                         ______________________________________                                                      T.sub.1     T.sub.1                                             Organ         Mn-EDTP-Ca  normal organs                                       ______________________________________                                        Heart         240         482                                                 Lung          413         585                                                 Fat           161         180                                                 Skeletal Muscle                                                                             260         411                                                 Renal Coster  101         342                                                 Renal Medulla  77         782                                                 Liver          43         260                                                 Spleen        200         473                                                 Pancreas      146         265                                                 Bladder       199         511                                                 Stomach       130         305                                                 Small Intestine                                                                             155         317                                                 Large Intestine                                                                             133         328                                                 ______________________________________                                    

By comparison according to Amer.J.Roentol. 143, 1226, the distributionof Gd DTPA in man at 30 minutes post-injection in milliseconds is:

                  TABLE 9                                                         ______________________________________                                        Organ           Pre T.sub.1                                                                           Post T.sub.1                                          ______________________________________                                        Fat             200     185                                                   Muscle          460     335                                                   Liver           350     195                                                   Spleen          560     285                                                   Kidneys         820     205                                                   ______________________________________                                    

The organ distribution pattern of Mn-EDTP-Ca is substantially differentfrom Gd-DTPA. It enters the hepatobiliary system resulting in asubstantial decrease in T₁ values of the liver, spleen, pancreas, andsmall and large intestines. Gd DTPA, being a vascular agent, is mainlycleared by the kidneys and does not substantially interact with thehepatobiliary system. Mn-EDTP-Ca also distributes to the heart. EKGstudies indicate that it does not disturb the function of the heart.

EXAMPLE 7

To 10 ml of water containing 5 ml of 1N sodium hydroxide is added 2.0 g(5 mmoles) of 1,4,7,10-tetraazacyclododecane - N,N',N'',N'''-tetraacetic acid. 1.3 g (5 mmoles) of GdCl₃ is added and thesuspension heated to 50° C. for 2 hours. Calcium chloride (1 mmole) isadded and the pH of the solution adjusted with 1N sodium hydroxide to6.5. The clear solution is filtered through a 0.2 micron filter forsterilization.

EXAMPLE 8

To 100 ml of water containing 10 g (100 mmoles) of N-methylglucamine isadded 19.7 g (50 mmoles) of diethylenetriamine-N,N',N'',N'''-pentaaceticacid. 13 g (50 moles) of GdCl₃ is added and the slurry stirred for 1hour at room temperature. Calcium ascorbate (3.9 g, 10 mmoles) is addedand the pH adjusted to 6.5 with 1N sodium hydroxide. The clear 500 mMsolution is filtered through a 0.2 micron filter for sterilization priorto use.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

We claim:
 1. A magnetic resonance imaging contrast medium comprising (a)a paramagnetic, physiologically compatible complex of anN-carboxymethylated-tetraazacyclododecane chelant and a paramagnetic ionof a lanthanide element of atomic number 57-70, and (b) atoxicity-reducing amount of labile calcium ions.
 2. A contrast medium asrecited in claim 1 wherein the toxicity-reducing calcium ions arederived from a source other than a salt of said chelant.
 3. A contrastmedium as recited in claim 2 wherein the toxicity-reducing calcium ionsare derived from an inorganic or organic calcium salt.
 4. A magneticresonance imaging contrast medium comprising (a) a paramagnetic,physiologically compatible salt of a physiologically compatible complexof an N-carboxymethylated-tetraazacyclododecane chelant and aparamagnetic ion of a lanthanide element of atomic number 57-70, and (b)a toxicity-reducing amount of labile calcium ions derived from a sourceother than a salt of said chelant.
 5. A contrast medium as recited inclaim 4 wherein the toxicity-reducing calcium ions are derived from aninorganic or organic calcium salt.
 6. A contrast medium as recited inclaim 5 wherein the paramagnetic salt is the calcium salt of thegadolinium complex1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid.
 7. Acontrast medium as recited in claim 1 wherein said chelant is1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid.
 8. Acontrast medium as recited in claim 1 wherein said lanthanide isgadolinium.
 9. A contrast medium as recited in claim 8 wherein saidchelant is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraaceticacid.
 10. A contrast medium as recited in claim 4 wherein said chelantis 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid.
 11. Acontrast medium as recited in claim 4 wherein said lanthanide isgadolinium.
 12. A contrast medium as recited in claim 11 wherein saidchelant is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraaceticacid.
 13. A magnetic resonance image-enhancing composition comprising amagnetic resonance image-enhancing effective amount of a contrast mediumas recited in any one of claims 1-12 inclusive, and a pharmaceuticallyacceptable diluent.
 14. In a method of magnetic resonance imaging inwhich a contrast medium comprising a physiologically compatibleparamagnetic complex of an N-carboxymethylated-tetraazacyclododecanechelant and a paramagnetic ion of a lanthanide element of atomic number57-50 is administered to a subject and a magnetic resonance image ofsaid subject is generated, the improvement comprising the presencewithin said contrast medium of a toxicity reducing amount of labilecalcium ions.
 15. A method as recited in claim 14 wherein thetoxicity-reducing calcium ions are derived from a source other than asalt of said chelant.
 16. A method as recited in claim 15 wherein thetoxicity-reducing calcium ions are derived from an inorganic or organiccalcium salt.
 17. A method as recited in claim 14 wherein said contrastmedium comprises (a) a paramagnetic, physiologically compatible salt ofa physiologically compatible complex of anN-carboxymethylated-tetraazacyclododecane chelant and a paramagnetic ionof a lanthanide element of atomic number 57-70 and (b) atoxicity-reducing amount of labile calcium ions derived from a sourceother than a salt of said chelant.
 18. A method as recited in claim 17wherein the toxicity-reducing calcium ions are derived from an inorganicor organic calcium salt.
 19. A method as recited in claim 14 whereinsaid chelant is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraaceticacid.
 20. A method as recited in claim 14 wherein said lanthanide isgadolinium.
 21. A method as recited in claim 19 wherein said lanthanideis gadolinium.